LOCUS       sequence-432387-        6975 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homologous recombination donor to insert halo tag at the N terminal 
            of the endogenous Ku70 locus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6975)
  AUTHORS   Mikhova M, Goff NJ, Janovic T, Heyza JR, Meek K, Schmidt JC
  TITLE     Single-molecule imaging reveals the kinetics of non-homologous 
            end-joining in living cells.
  JOURNAL   Nat Commun. 2024 Nov 23;15(1):10159. doi: 
            10.1038/s41467-024-54545-y.
  PUBMED    39578493
REFERENCE   2  (bases 1 to 6975)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2024-11-23"; volume: "15(1):10159. doi"; pages: " 
            10.1038/s41467-024-54545-y"
COMMENT     Sequence Label: N-terminal Halo-Ku70
FEATURES             Location/Qualifiers
     source          1..6975
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     254..271
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  405..629
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(696..1229)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(1418..1423,1424..1440,1441..1446))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    1954..2002
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     2027..2046
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(2138..2247)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        2271..2362
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     2287..2304
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             2870..2935
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    2936..2969
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2978..3868
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             3881..3901
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     polyA_signal    4359..4493
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4396..4415)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4450..4469
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  4522..4687
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      4871..5325
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4958..4977)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5167..5188
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5351..5455
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5456..5524,5525..6316)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5674..5693)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6487..6975,1..100)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
       61 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
      121 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
      181 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
      241 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
      301 tctccttacg catctgtgcg gtatttcaca ccgcatagac cagccgcgta acctggcaaa
      361 atcggttacg gttgagtaat aaatggatgc cctgcgtaag cgggtgtggg cggacaataa
      421 agtcttaaac tgaacaaaat agatctaaac tatgacaata aagtcttaaa ctagacagaa
      481 tagttgtaaa ctgaaatcag tccagttatg ctgtgaaaaa gcatactgga cttttgttat
      541 ggctaaagca aactcttcat tttctgaagt gcaaattgcc cgtcgtatta aagaggggcg
      601 tggccaaggg catggtaaag actatattcg cggcgttgtg acaatttacc gaacaactcc
      661 gcggccggga agccgatctc ggcttgaacg aattgttagg tggcggtact tgggtcgata
      721 tcaaagtgca tcacttcttc ccgtatgccc aactttgtat agagagccac tgcgggatcg
      781 tcaccgtaat ctgcttgcac gtagatcaca taagcaccaa gcgcgttggc ctcatgcttg
      841 aggagattga tgagcgcggt ggcaatgccc tgcctccggt gctcgccgga gactgcgaga
      901 tcatagatat agatctcact acgcggctgc tcaaacttgg gcagaacgta agccgcgaga
      961 gcgccaacaa ccgcttcttg gtcgaaggca gcaagcgcga tgaatgtctt actacggagc
     1021 aagttcccga ggtaatcgga gtccggctga tgttgggagt aggtggctac gtctccgaac
     1081 tcacgaccga aaagatcaag agcagcccgc atggatttga cttggtcagg gccgagccta
     1141 catgtgcgaa tgatgcccat acttgagcca cctaactttg ttttagggcg actgccctgc
     1201 tgcgtaacat cgttgctgct gcgtaacatc gttgctgctc cataacatca aacatcgacc
     1261 cacggcgtaa cgcgcttgct gcttggatgc ccgaggcata gactgtacaa aaaaacagtc
     1321 ataacaagcc atgaaaaccg ccactgcgcc gttaccaccg ctgcgttcgg tcaaggttct
     1381 ggaccagttg cgtgagcgca tacgctactt gcattacagt ttacgaaccg aacaggctta
     1441 tgtcaactgg gttcgtgcct tcatccgttt ccacggtgtg cgtcacccgg caaccttggg
     1501 cagcagcgaa gtcgaggcat ttctgtcctg gctggcgaac gagcgcaagg tttcggtctc
     1561 cacgcatcgt caggcattgg cggccttgct gttcttctac ggcaaggtgc tgtgcacgga
     1621 tctgcccttg cttcaggaga tcggtagacc tcggccgtcg cggcgcttgc cggtggtgct
     1681 gaccccggat gaagtggttc gcatcctcgg ttttctggaa ggcgagcatc gtttgttcgc
     1741 ccaggactct agctatagtt ctagtggttg gctacgtacc cgtagtggct atggcagggc
     1801 ttgccgcccc gacgttggct gcgagccctg ggccttcacc cgaacttggg ggttggggtg
     1861 gggaaaagga agaaacgcgg gcgtattggt cccaatgggg tctcggtggg gtatcgacag
     1921 agtgccagcc ctgggaccga accccgcgtt tatgaacaaa cgacccaaca cccgtgcgtt
     1981 ttattctgtc tttttattgc cgtcatagcg cgggttcctt ccggtattgt ctccttccgt
     2041 gtttcagtta gcctccccca tctcccggta ccgcatgcta tgcatcagct gctagcacca
     2101 tggctcgaga tcccgggtga tcaagtcttc gtcgagtgat tgtaaataaa atgtaattta
     2161 cagtatagta ttttaattaa tatacaaatg atttgataat aattcttatt taactataat
     2221 atattgtgtt gggttgaatt aaaggtccgt atactccgga atattaatag atcatggaga
     2281 taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc gtaacagttt
     2341 tgtaataaaa aaacctataa atattccgga ttattcatac cgtcccacca tcgggcgcgg
     2401 atcccggtcc gaagcgcgcg gaattcaaag gcctacgtcg acgagctcac tagtcgcggc
     2461 cgctttcgaa tctagagcct gcagtctcga caagcttgtc gagaagtact agaggatcat
     2521 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     2581 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt ggccgccgcc ggccctgaaa
     2641 cgtgagggat agctgagatg aggcagctac tgggatggcc cccatgcgca tttacatgca
     2701 gtccgactgc cgagctttcg aggcagcagg atttaccgtc cacattcctc actactaacc
     2761 aagcttttag aacagatctc acaagaacct agaggtcggt attttttcga tttaaatttg
     2821 cctgttactg acgttaacgt ctttcgccta gtgagcagta gccaacatgg actacaaaga
     2881 ccatgacggt gattataaag atcatgatat cgattacaag gatgacgatg acaagataac
     2941 ttcgtataat gtatgctata cgaagttatc cggtaccatg gcagaaatcg gtactggctt
     3001 tccattcgac ccccattatg tggaagtcct gggcgagcgc atgcactacg tcgatgttgg
     3061 tccgcgcgat ggcacccctg tgctgttcct gcacggtaac ccgacctcct cctacgtgtg
     3121 gcgcaacatc atcccgcatg ttgcaccgac ccatcgctgc attgctccag acctgatcgg
     3181 tatgggcaaa tccgacaaac cagacctggg ttatttcttc gacgaccacg tccgcttcat
     3241 ggatgccttc atcgaagccc tgggtctgga agaggtcgtc ctggtcattc acgactgggg
     3301 ctccgctctg ggtttccact gggccaagcg caatccagag cgcgtcaaag gtattgcatt
     3361 tatggagttc atccgcccta tcccgacctg ggacgaatgg ccagaatttg cccgcgagac
     3421 cttccaggcc ttccgcacca ccgacgtcgg ccgcaagctg atcatcgatc agaacgtttt
     3481 tatcgagggt acgctgccga tgggtgtcgt ccgcccgctg actgaagtcg agatggacca
     3541 ttaccgcgag ccgttcctga atcctgttga ccgcgagcca ctgtggcgct tcccaaacga
     3601 gctgccaatc gccggtgagc cagcgaacat cgtcgcgctg gtcgaagaat acatggactg
     3661 gctgcaccag tcccctgtcc cgaagctgct gttctggggc accccaggcg ttctgatccc
     3721 accggccgaa gccgctcgcc tggccaaaag cctgcctaac tgcaaggctg tggacatcgg
     3781 cccgggtctg aatctgctgc aagaagacaa cccggacctg atcggcagcg agatcgcgcg
     3841 ctggctgtcg acgctcgaga tttccggcga gccaaccact gaggatctgt actttcagag
     3901 cgataacgcg atcgcttccg gtacctcagg gtgggagtca tattacaaaa ccgagggcga
     3961 tgaagaagca gaggaagaac aagaagagaa ccttgaagca agtggtaagt gacttcagca
     4021 tgtagtgcca ttcggtgtgt ggaagaaaga ccttccctgc ctatctctgc tggatggact
     4081 ttgctgtttg atggcctgcc cttctcctcc cagataattc tgaaattctt ttcttccttt
     4141 gaacttcgca gagctaccct aggctcttat atgcagatct tactctagtg tgccctaagt
     4201 tttttttgga cactggtagt ttgacatctt ttggtccctg ataaatggcc atgaccatag
     4261 gtattttttg tcatcttaag tggttcaggt gagacccttc tcatccagga aagtagtgtt
     4321 accattccag gacctcagtg taaaaatgcc cccaccctaa cttgtttatt gcagcttata
     4381 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4441 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atctgatcac
     4501 tgcttgagcc taggagatcc gaaccagata agtgaaatct agttccaaac tattttgtca
     4561 tttttaattt tcgtattagc ttacgacgct acacccagtt cccatctatt ttgtcactct
     4621 tccctaaata atccttaaaa actccatttc cacccctccc agttcccaac tattttgtcc
     4681 gcccacagcg gggcattttt cttcctgtta tgtttttaat caaacatcct gccaactcca
     4741 tgtgacaaac cgtcatcttc ggctactttt tctctgtcac agaatgaaaa tttttctgtc
     4801 atctcttcgt tattaatgtt tgtaattgac tgaatatcaa cgcttatttg cagcctgaat
     4861 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4921 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4981 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     5041 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     5101 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5161 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     5221 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     5281 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     5341 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5401 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5461 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5521 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5581 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5641 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5701 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5761 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5821 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5881 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5941 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6001 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6061 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6121 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     6181 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6241 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6301 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     6361 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6421 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6481 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6541 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6601 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6661 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6721 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6781 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6841 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6901 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6961 gagggagctt ccagg
//
